Both focal and diffuse brain injury pathologies have been suggested to be linked to destructive calcium-mediated processes. One likely mediator of such intracellular auto-destructive processes is the calpain family of neutral proteases. Calpains are calcium-activated and are capable of effecting the limited proteolysis of many intracellular proteins, allowing for transient or permanent alterations in cellular function or structural integrity. The central hypothesis of Project 2 is that traumatic brain injury leads to activation of calpains which subsequently degrade neurofilament proteins, structural elements which may play an important role in resistance to and recovery from injury. The long range goals of the project are twofold: 1) to identify the distinct features of calpain activation and NF damage for white matter and gray matter injuries in order to develop more effective, targeted therapeutic strategies for calpain inhibition following brain injury, and 2) to identify the potential roles for NFs in modulating the initial impact of mechanical trauma and in influencing the progression of trauma-induced neuropathology. To achieve these goals, we propose in Aim 1 to establish the patterns of activation of calpains I and II and associated NF damage in brain-injured humans and use this information to validate the well-characterized rat model of lateral fluid percussion injury, which mimics the mixture of gray and white matter pathology commonly seen in humans. In Aim 2, we propose to study, in the mouse, gray matter and white matter pathology in isolation, using the controlled cortical impact (CCI) model and the optic nerve stretch model. This will allow us to directly compare the time course of calpain activation and NF damage in gray vs. white matter regions to reestablish targeting strategies for therapy. The proteolytic events observed in mouse optic nerve injury will then be confirmed using a model of diffuse axonal injury (DAI) in the pig which closely reproduces human DAI pathology. In Aim 3 we will return to the mouse CCI model to exploit the availability of genetically altered (transgenic and knockout) mice to mechanistically explore the role of NFs in trauma-induced pathology, behavioral dysfunction and gene expression. We hope that increased understanding of the role of NFs in trauma and their relationship to calpain-mediated pathology may lead to improve therapies aimed at inhibition of calpain and the development of novel therapeutic approaches targeting the neurofilamentous cytoskeleton.